Vikas P. Sukhatme

Excess placental soluble fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia

Preeclampsia, a syndrome affecting 5% of pregnancies, causes substantial maternal and fetal morbidity and mortality. The pathophysiology of preeclampsia remains largely unknown. It has been hypothesized that placental ischemia is an early event, leading to placental production of a soluble factor or factors that cause maternal endothelial dysfunction, resulting in the clinical findings of hypertension, proteinuria, and edema. Here, we confirm that placental soluble fms-like tyrosine kinase 1 (sFlt1), an antagonist of VEGF and placental growth factor (PlGF), is upregulated in preeclampsia, leading to increased systemic levels of sFlt1 that fall after delivery. We demonstrate that increased circulating sFlt1 in patients with preeclampsia is associated with decreased circulating levels of free VEGF and PlGF, resulting in endothelial dysfunction in vitro that can be rescued by exogenous VEGF and PlGF. Additionally, VEGF and PlGF cause microvascular relaxation of rat renal arterioles in vitro that is blocked by sFlt1. Finally, administration of sFlt1 to pregnant rats induces hypertension, proteinuria, and glomerular endotheliosis, the classic lesion of preeclampsia. These observations suggest that excess circulating sFlt1 contributes to the pathogenesis of preeclampsia.

Soluble endoglin contributes to the pathogenesis of preeclampsia.

Preeclampsia is a pregnancy-specific hypertensive syndrome that causes substantial maternal and fetal morbidity and mortality. Maternal endothelial dysfunction mediated by excess placenta-derived soluble VEGF receptor 1 (sVEGFR1 or sFlt1) is emerging as a prominent component in disease pathogenesis. We report a novel placenta-derived soluble TGF-β coreceptor, endoglin (sEng), which is elevated in the sera of preeclamptic individuals, correlates with disease severity and falls after delivery. sEng inhibits formation of capillary tubes in vitro and induces vascular permeability and hypertension in vivo. Its effects in pregnant rats are amplified by coadministration of sFlt1, leading to severe preeclampsia including the HELLP (hemolysis, elevated liver enzymes, low platelets) syndrome and restriction of fetal growth. sEng impairs binding of TGF-β1 to its receptors and downstream signaling including effects on activation of eNOS and vasodilation, suggesting that sEng leads to dysregulated TGF-β signaling in the vasculature. Our results suggest that sEng may act in concert with sFlt1 to induce severe preeclampsia.

A zinc finger-encoding gene coregulated with c-fos during growth and differentiation, and after cellular depolarization

Egr-1 is an early growth response gene that displays fos-like induction kinetics in fibroblasts, epithelial cells, and lymphocytes following mitogenic stimulation. Sequence analysis of murine Egr-1 cDNA predicts a protein with three DNA binding zinc fingers. The human EGR1 gene maps to chromosome 5 (bands 5q23-31). Egr-1 mRNA increases dramatically during cardiac and neural cell differentiation, and following membrane depolarization both in vitro and in vivo. Thus, Egr-1 and c-fos are often coregulated with strikingly similar kinetics. These results, in conjunction with the Egr-1 primary structure, suggest that Egr-1 may function as a transcriptional regulator in diverse biological processes.

Combined effects of angiostatin and ionizing radiation in antitumour therapy

Angiogenesis, the formation of new capillaries from pre-existing vessels, is essential for tumour progression. Angiostatin, a proteolytic fragment of plasminogen that was first isolated from the serum and urine of tumour-bearing mice, inhibits angiogenesis and thereby growth of primary and metastatic,, tumours. Radiotherapy is important in the treatment of many human cancers, but is often unsuccessful because of tumour cell radiation resistance,. Here we combine radiation with angiostatin to target tumour vasculature that is genetically stable and therefore less likely to develop resistance. The results show an antitumour interaction between ionizing radiation and angiostatin for four distinct tumour types, at doses of radiation that are used in radiotherapy. The combination produced no increase in toxicity towards normal tissue. In vitro studies show that radiation and angiostatin have combined cytotoxic effects on endothelial cells, but not tumour cells. In vivo studies show that these agents, in combination, target the tumour vasculature. Our results provide support for combining ionizing radiation with angiostatin to improve tumour eradication without increasing deleterious effects.

Endostatin induces endothelial cell apoptosis.

Endostatin, a carboxyl-terminal fragment of collagen XVIII, has been shown to regress tumors in mice. In this study, we have analyzed the mechanism of endostatin action on endothelial cells and nonendothelial cells. Endostatin treatment of cow pulmonary artery endothelial cells caused apoptosis, as demonstrated by three methods, annexin V-fluorescein isothiocyanate staining, caspase 3, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling assay. Moreover, addition of endostatin led to a marked reduction of the Bcl-2 and Bcl-XLanti-apoptotic protein, whereas Bax protein levels were unaffected. These effects were not seen in several nonendothelial cells. Collectively, these findings provide important mechanistic insight into endostatin action.

Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors.

Publisher Summary This chapter discusses the early growth response protein (Egr) gene family and its most extensively characterized member, Egr-1, first identified as an immediate-early gene responsive to growth factors and various differentiation cues, later confirmed to be a transcriptional regulatory protein. During development, a single Egr-1 transcript is predominantly expressed in cortex, midbrain, and cerebellum; in bone, cartilage, and muscle; and at several sites of epithelial-mesenchymal interactions. In addition, Egr-1 is expressed in diverse physiological contexts in particular cell types. The broad spectrum of extracellular stimuli that induce Egr-1 can be roughly subgrouped into four categories: (1) mitogens, (2) developmental or differentiation cues, (3) tissue or radiation injury, and (4) signals that cause neuronal excitation. Strong evidence for a role for Egr-1 in proliferation also comes, from the studies, with mouse skeletal muscle So18 cells. Although Egr-1 message was induced in response to mitogenic stimuli, differentiative stimuli (insulin), and other agents that caused neither proliferation nor differentiation, Egr-1 protein could be detected only in response to the mitogenic cues.

Canstatin, a Novel Matrix-derived Inhibitor of Angiogenesis and Tumor Growth

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the α2 chain of type IV collagen, was produced as a recombinant molecule inEscherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.

Excess Circulating Angiopoietin-2 May Contribute to Pulmonary Vascular Leak in Sepsis in Humans

Background Acute respiratory distress syndrome (ARDS) is a devastating complication of numerous underlying conditions, most notably sepsis. Although pathologic vascular leak has been implicated in the pathogenesis of ARDS and sepsis-associated lung injury, the mechanisms promoting leak are incompletely understood. Angiopoietin-2 (Ang-2), a known antagonist of the endothelial Tie-2 receptor, was originally described as a naturally occurring disruptor of normal embryonic vascular development otherwise mediated by the Tie-2 agonist angiopoietin-1 (Ang-1). We hypothesized that Ang-2 contributes to endothelial barrier disruption in sepsis-associated lung injury, a condition involving the mature vasculature. Methods and Findings We describe complementary human, murine, and in vitro investigations that implicate Ang-2 as a mediator of this process. We show that circulating Ang-2 is significantly elevated in humans with sepsis who have impaired oxygenation. We then show that serum from these patients disrupts endothelial architecture. This effect of sepsis serum from humans correlates with measured Ang-2, abates with clinical improvement, and is reversed by Ang-1. Next, we found that endothelial barrier disruption can be provoked by Ang-2 alone. This signal is transduced through myosin light chain phosphorylation. Last, we show that excess systemic Ang-2 provokes pulmonary leak and congestion in otherwise healthy adult mice. Conclusions Our results identify a critical role for Ang-2 in disrupting normal pulmonary endothelial function.

Hemodialysis Vascular Access Dysfunction: A Cellular and Molecular Viewpoint

Hemodialysis vascular access dysfunction is a major cause of morbidity and hospitalization in the hemodialysis population. The major cause of hemodialysis vascular access dysfunction is venous stenosis as a result of neointimal hyperplasia. Despite the magnitude of the clinical problem, however, there has been a paucity of novel therapeutic interventions in this field. This is in marked contrast to a recent plethora of targeted interventions for the treatment of arterial neointimal hyperplasia after coronary angioplasty. The reasons for this are two-fold. First there has been a relative lack of cellular and molecular research that focuses on venous neointimal hyperplasia in the specific setting of hemodialysis vascular access. Second, there have been inadequate efforts by the nephrology community to translate the recent advances in molecular and interventional cardiology into therapies for hemodialysis vascular access. This review therefore (1) briefly examines the different forms of hemodialysis vascular access that are available, (2) describes the pathology and pathogenesis of hemodialysis vascular access dysfunction in both polytetrafluoroethylene grafts and native arteriovenous fistulae, (3) reviews recent concepts about the pathogenesis of vascular stenosis that could potentially be applied in the setting of hemodialysis vascular access dysfunction, (4) summarizes novel experimental and clinical therapies that could potentially be used in the setting of hemodialysis vascular access dysfunction, and, finally, (5) offers some broad guidelines for future innovative translational and clinical research in this area that hopefully will reduce the huge clinical morbidity and economic costs that are associated with this condition.

The von Hippel-Lindau tumor suppressor gene product interacts with Sp1 to repress vascular endothelial growth factor promoter activity

The von Hippel-Lindau tumor suppressor gene (VHL) has a critical role in the pathogenesis of clear-cell renal cell carcinoma (RCC), as VHL mutations have been found in both von Hippel-Lindau disease-associated and sporadic RCCs. Recent studies suggest that vascular endothelial growth factor (VEGF) mRNA is upregulated in RCC- and von Hippel-Lindau disease-associated tumors. We have therefore assessed the effect of the VHL gene product on VEGF expression. VEGF promoter-luciferase constructs were transiently cotransfected with a wild-type VHL (wt-VHL) vector in several cell lines, including 293 embryonic kidney and RCC cell lines. wt-VHL protein inhibited VEGF promoter activity in a dose-dependent manner up to 5- to 10-fold. Deletion analysis defined a 144-bp region of the VEGF promoter necessary for VHL repression. This VHL-responsive element is GC rich and specifically binds the transcription factor Sp1 in crude nuclear extracts. In Drosophila cells, cotransfected VHL represses Sp1-mediated activation but not basal activity of the VEGF promoter. We next demonstrated in coimmunoprecipitates that VHL and Sp1 were part of the same complex and, by using a glutathione-S-transferase-VHL fusion protein and purified Sp1, that VHL and Sp1 directly interact. Furthermore, endogenous VEGF mRNA levels were suppressed in permanent RCC cell lines expressing wt-VHL, and nuclear run-on studies indicated that VHL regulation of VEGF occurs at least partly at the transcriptional level. These observations support a new mechanism for VHL-mediated transcriptional repression via a direct inhibitory action on Sp1 and suggest that loss of Sp1 inhibition may be important in the pathogenesis of von Hippel-Lindau disease and RCC.